Orientational Control over Nitrite Reductase on Modified Gold Electrode and Its Effects on the Interfacial Electron Transfer

Abstract

Recently, studies have been reported in which fluorescently labeled redox proteins have been studied with a combination of spectroscopy and electrochemistry. In order to understand the effect of the dye on the protein-electrode interaction, voltammetry and surface analysis have been performed on protein films of dye-labeled and unlabeled forms of a cysteine-surface variant (L93C) and the wild type (wt) of the copper containing nitrite reductase (NiR) from Alcaligenes faecalis S6. The protein has been adsorbed onto gold electrodes modified with self-assembled monolayers (SAMs) made up of 6-mercaptohexanol (6-OH) and mixtures of various octanethiols. Electrochemical and surface-analytical techniques were utilized to explore the influence of the SAM composition on wt and L93C NiR enzyme activity and the orientation of the enzyme molecules with respect to the electrode/SAM. The unlabeled L93C NiR enzyme is only electroactive on mixed SAMs composed of positive 8-aminooctanethiol (8-NH(2)) and 8-mercaptooctanol (8-OH). No enzymatic activity is observed on SAMs consisting of pure 6-OH, 8-OH, or pure 8-NH(2). Modification of L93C NiR with the ATTO 565 dye resulted in enzymatic activity on SAMs of 6-OH, but not on SAMs of 8-OH. Quartz crystal microbalance with dissipation measurements show that well-ordered and rigid protein films (single orientation of the protein) are formed when NiR is electroactive. By contrast, electrode-NiR combinations for which no electrochemical activity is observed still have NiR adsorbed on the surfaces, but a less-structured and water-rich film is formed. For the unlabeled L93C NiR, bilayer formation is observed, suggesting that the Cys93 residue is orientated away from the surface and able to form disulfide bridges to a second layer of L93C NiR. The results indicate that interfacial electron transfer is only possible if the negatively charged surface patch surrounding the electron-entry site of NiR is directed toward the electrode. This can be achieved either by introducing positive charges in the SAM or, when the SAM does not carry a charge, by labeling the enzyme with an ATTO 565 dye, which has some hydrophobic character, close to the electron entry site of the NiR.

Controlling Redox Enzyme Orientation at Planar Electrodes

“…Mixed SAMs present stronger overall polarization than pure SAMs as a consequence of antiparallel orientation of dipoles within the SAM. In case of electrostatic binding of enzymes, this polarization induces an enhanced amount of enzyme loading [183]. Many studies dedicated to enzymatic electrocatalysis use mixed SAMs, although the partition of the thiols in such mixed layers is not straightforward, and a detailed relationship between the SAM mixed chemistry and the enzyme activity is rarely provided.…”

Facilitating electron transfer in bioelectrocatalytic systems

“…Red parts of the SAM on the lef t scheme demonstrate dif f erence in the composition of the layer. Reprinted with the permission f rom 135 . Copyright © 2011 American Chemical Society.…”

Probing redox reaction of azurin protein immobilized on hydroxyl-terminated self-assembled monolayers with different lengths

“…Third, SAM-modified electrodes pave the way for protein investigation with high sensitivity in trace amounts [4]. Lastly, it is possible to have a control-over-orientation of immobilized protein, by using different functional groups on the SAM [5].…”